Injectable botulinum toxin formulations

ABSTRACT

This invention provides novel injectable compositions comprising botulinum toxin that may be administered to a subject for various therapeutic, aesthetic and/or cosmetic purposes. The injectable compositions contemplated by the invention exhibit one or more advantages over conventional botulinum toxin formulations, including reduced antigenicity, a reduced tendency to undergo unwanted localized diffusion following injection, increased duration of clinical efficacy or enhanced potency relative, faster onset of clinical efficacy, and/or improved stability.

RELATED PATENT APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 13/141,935, filed Jul. 19, 2011, which in turn is filed under35 U.S.C. § 371 as the U.S. national stage application of InternationalPatent Application No. PCT/US2009/069576, filed Dec. 28, 2009, whichclaims the benefit of priority under 35 U.S.C. § 119 to U.S. ProvisionalPatent Application No. 61/142,063, filed Dec. 31, 2008, the contents ofeach of which are incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Feb. 5, 2010, is named20105131.txt, and is 4,114 bytes in size.

FIELD OF THE INVENTION

This invention relates to novel injectable compositions comprisingbotulinum toxin that may be administered to a subject for varioustherapeutic, aesthetic and/or cosmetic purposes.

BACKGROUND OF THE INVENTION

Skin protects the body's organs from external environmental threats andacts as a thermostat to maintain body temperature. It consists ofseveral different layers, each with specialized functions. The majorlayers include the epidermis, the dermis and the hypodermis. Theepidermis is a stratifying layer of epithelial cells that overlies thedermis, which consists of connective tissue. Both the epidermis and thedermis are further supported by the hypodermis, an internal layer ofadipose tissue.

The epidermis, the topmost layer of skin, is only 0.1 to 1.5 millimetersthick (Inlander, Skin, New York, N.Y.: People's Medical Society, 1-7(1998)). It consists of keratinocytes and is divided into several layersbased on their state of differentiation. The epidermis can be furtherclassified into the stratum corneum and the viable epidermis, whichconsists of the granular melphigian and basal cells. The stratum corneumis hygroscopic and requires at least 10% moisture by weight to maintainits flexibility and softness. The hygroscopicity is attributable in partto the water-holding capacity of keratin. When the horny layer loses itssoftness and flexibility it becomes rough and brittle, resulting in dryskin.

The dermis, which lies just beneath the epidermis, is 1.5 to 4millimeters thick. It is the thickest of the three layers of the skin.Most of the skin's structures, including sweat and oil glands (whichsecrete substances through openings in the skin called pores, orcomedos), hair follicles, nerve endings, and blood and lymph vessels arefound in the dermis (Inlander, Skin, New York, N.Y.: People's MedicalSociety, 1-7 (1998)). However, the main components of the dermis arecollagen and elastin.

The hypodermis is the deepest layer of the skin. It acts both as aninsulator for body heat conservation and as a shock absorber for organprotection (Inlander, Skin, New York, N.Y.: People's Medical Society,1-7 (1998)). In addition, the hypodermis also stores fat for energyreserves. The pH of skin is normally between 5 and 6. This acidity isdue to the presence of amphoteric amino acids, lactic acid, and fattyacids from the secretions of the sebaceous glands. The term “acidmantle” refers to the presence of the water-soluble substances on mostregions of the skin. The buffering capacity of the skin is due in partto these secretions stored in the skin's horny layer.

Wrinkles, one of the telltale signs of aging, can be caused bybiochemical, histological, and physiologic changes that accumulate fromenvironmental damage to the skin. (Benedetto, “International Journal ofDermatology,” 38:641-655 (1999)). In addition, there are other secondaryfactors that can cause characteristic folds, furrows, and creases offacial wrinkles (Stegman et al., The Skin of the Aging Face CosmeticDermatological Surgery, 2^(nd) ed., St. Louis, Mo.: Mosby Year Book:5-15 (1990)). These secondary factors include the constant pull ofgravity, frequent and constant positional pressure on the skin (e.g.,during sleep), and repeated facial movements caused by the contractionof facial muscles (Stegman et al., The Skin of the Aging Face CosmeticDermatological Surgery, 2^(nd) ed., St. Louis, Mo.: Mosby Year Book:5-15 (1990)).

Different techniques have been utilized in order to potentially mollifysome of the signs of aging. These techniques range from facialmoisturizers containing alpha hydroxy acids and retinol to surgicalprocedures and injections of neurotoxins. For example, in 1986, Jean andAlastair Carruthers, a husband and wife team consisting of an ocuplasticsurgeon and a dermatologist, developed a method of using the type A formof botulinum toxin for treatment of movement-associated wrinkles in theglabella area (Schantz and Scott, In Lewis G. E. (Ed) Biomedical Aspectsof Botulinum, New York: Academic Press, 143-150 (1981)). The Carruthers'use of the type A form of botulinum toxin for the treatment of wrinklesled to the seminal publication of this approach in 1992 (Schantz andScott, In Lewis G. E. (Ed) Biomedical Aspects of Botulinum, New York:Academic Press, 143-150 (1981)). By 1994, the same team reportedexperiences with other movement-associated wrinkles on the face (Scott,Ophthalmol, 87:1044-1049 (1980)). This in turn led to the birth of theera of cosmetic treatment using the type A form of botulinum toxin.

The type A form of botulinum toxin is reported to be the most lethalnatural biological agent known to man. Spores of C. botulinum are foundin soil and can grow in improperly sterilized and sealed foodcontainers. Botulism, which may be fatal, may be caused by the ingestionof the bacteria. Botulinum toxin acts to produce paralysis of muscles bypreventing synaptic transmission by inhibiting the release ofacetylcholine across the neuromuscular junction, and is thought to actin other ways as well. Its action essentially blocks signals thatnormally would cause muscle spasms or contractions, resulting inparalysis. During the last decade, botulinum toxin's muscle paralyzingactivity has been harnessed to achieve a variety of therapeutic effects.Controlled administration of botulinum toxin has been used to providemuscle paralysis to treat a variety of medical conditions, for example,neuromuscular disorders characterized by hyperactive skeletal muscles.Conditions that have been treated with botulinum toxin includehemifacial spasm, adult onset spasmodic torticollis, anal fissure,blepharospasm, cerebral palsy, cervical dystonia, migraine headaches,strabismus, temporomandibular joint disorder, and various types ofmuscle cramping and spasms. More recently, the muscle-paralyzing effectsof botulinum toxin have been applied to therapeutic and cosmetic facialapplications such as treatment of wrinkles, frown lines, and otherresults of spasms or contractions of facial muscles.

In addition to the type A form of botulinum toxin, there are seven otherserologically distinct forms of botulinum toxin that are also producedby the gram-positive bacteria Clostridium botulinum. Of these eightserologically distinct types of botulinum toxin, the seven that cancause paralysis have been designated botulinum toxin serotypes A, B, C,D, E, F and G. Each of these is distinguished by neutralization withtype-specific antibodies. The molecular weight of each of the botulinumtoxin proteins is about 150 kD. Due to the molecule size and molecularstructure of botulinum toxin, it cannot cross stratum corneum and themultiple layers of the underlying skin architecture. The differentserotypes of botulinum toxin vary in the effect and in the severity andduration of the paralysis they evoke in different animal species. Forexample, in rats, it has been determined that botulinum toxin type A is500 times more potent than botulinum toxin type B, as measured by therate of paralysis. Additionally, botulinum toxin type B has beendetermined to be non-toxic in primates at a dose of 480 U/kg, about 12times the primate LD₅₀ for type A.

As released by Clostridium botulinum bacteria, botulinum toxin is acomponent of a toxin complex containing the approximately 150 kDbotulinum toxin protein molecule along with associated non-toxinproteins. These endogenous non-toxin proteins are believed to include afamily of hemagglutinin proteins, as well as non-hemagglutinin protein.The non-toxin proteins have been reported to stabilize the botulinumtoxin molecule in the toxin complex and protect it against denaturationby digestive acids when toxin complex is ingested. Thus, the non-toxinproteins of the toxin complex protect the activity of the botulinumtoxin and thereby enhance systemic penetration when the toxin complex isadministered via the gastrointestinal tract. Additionally, it isbelieved that some of the non-toxin proteins specifically stabilize thebotulinum toxin molecule in blood.

The presence of non-toxin proteins in the toxin complexes typicallycauses the toxin complexes to have molecular weights that are greaterthan that of the bare botulinum toxin molecule, which is about 150 kD,as previously stated. For example, Clostridium botulinum bacteria canproduce botulinum type A toxin complexes that have molecular weights ofabout 900 kD, 500 kD or 300 kD. Botulinum toxin types B and C areproduced as complexes having a molecular weight of about 700 kD or about500 kD. Botulinum toxin type D is produced as complexes having molecularweights of about 300 kD or 500 kD. Botulinum toxin types E and F areonly produced as complexes having a molecular weight of about 300 kD.

To provide additional stability to botulinum toxin, the toxin complexesare conventionally stabilized by combining the complexes with albuminduring manufacturing. For example, BOTOX® (Allergan, Inc., Irvine,Calif.) is a botulinum toxin-containing formulation that contains 100 Uof type A botulinum toxin with accessory proteins, 0.5 milligrams ofhuman albumin, and 0.9 milligrams of sodium chloride. The albumin servesto bind and to stabilize toxin complexes in disparate environments,including those associated with manufacturing, transportation, storage,and administration.

Typically, the botulinum toxin is administered to patients by carefullycontrolled injections of compositions containing botulinum toxin complexand albumin. However, there are several problems associated with thisapproach. Not only are the injections painful, but typically largesubdermal wells of toxin are locally generated around the injectionsites, in order to achieve the desired therapeutic or cosmetic effect.The botulinum toxin may migrate from these subdermal wells to causeunwanted paralysis in surrounding areas of the body. This problem isexacerbated when the area to be treated is large and many injections oftoxin are required to treat the area. Moreover, because the injectedtoxin complexes contain non-toxin proteins and albumin that stabilizethe botulinum toxin and increase the molecular weight of the toxincomplex, the toxin complexes have a long half-life in the body and maycause an undesirable antigenic response in the patient. For example,some patients will, over time, develop an allergy to the albumin used asa stabilizer in current commercial formulations. Also, the toxincomplexes may induce the immune system of the patient to formneutralizing antibodies, so that larger amounts of toxin are required insubsequent administrations to achieve the same effect. When thishappens, subsequent injections must be carefully placed so that they donot release a large amount of toxin into the bloodstream of the patient,which could lead to fatal systemic poisoning, especially since thenon-toxin proteins and albumin stabilize the botulinum toxin in blood.

In view of the drawbacks associated with current botulinum toxinformulations, it would be highly desirable to have an injectablebotulinum toxin formulation that is efficacious and stable, but exhibitsreduced antigenicity and a lower tendency to diffuse locally afterinjection. It would also be desirable to use such a botulinum toxinformulation for various therapeutic, aesthetic and/or cosmetic purposes.

SUMMARY OF THE INVENTION

This invention provides injectable compositions comprising botulinumtoxin non-covalently associated with a positively charged carriermolecule. In preferred embodiments, the compositions of the inventionpossess one or more advantages over conventional commercial botulinumtoxin formulations, such as BOTOX® or MYOBLOC®. For instance, in certainembodiments, the compositions may exhibit one or more advantages overconventional injectable botulinum formulations, including reducedantigenicity, a reduced tendency to undergo diffusion into surroundingtissue following injection, increased duration of clinical efficacy orenhanced potency relative to conventional botulinum toxin formulations,faster onset of clinical efficacy, and/or improved stability.

One aspect of this invention is the recognition that certain non-nativemolecules (i.e., molecules not found in botulinum toxin complexesobtained from Clostridium botulinum bacteria) can be added to botulinumtoxin, botulinum toxin complexes, and in particular reduced botulinumtoxin complexes (as defined herein), to improve toxin diffusion throughtissues. The non-native molecules associate non-covalently with thetoxin and act as penetration enhancers that improve the ability of thetoxin to reach target structures after injection. Furthermore, thenon-native molecules may increase the stability of the toxin prior toand after injection. By way of example, the penetration enhancers may bepositively charged carriers, such as cationic peptides, which have noinherent botulinum-toxin-like activity and which also contain one ormore protein transduction domains as described herein.

Another embodiment of this invention is to provide a compositioncomprising botulinum toxin, a botulinum toxin complex (or a reducedprotein botulinum toxin complex including just the 150 kD neurotoxinitself or the neurotoxin with some, but not all, of the native complexproteins) and a positively charged carrier.

The invention further relates to a method for producing a biologiceffect by injecting an effective amount of the compositions of thisinvention to a subject or patient in need of such treatment. Thebiologic effect may include, for example, muscle paralysis, reduction ofhypersecretion or sweating, treatment of neurologic pain or migraineheadache, management of rhinitis or sinusitis, treatment of hyperactivebladder, reduction of muscle spasms, prevention or reduction of acne,reduction or enhancement of an immune response, reduction of wrinkles,or prevention or treatment of various other disorders.

This invention also provides kits for preparing formulations containinga botulinum toxin, a botulinum toxin complex, or a reduced proteinbotulinum toxin complex and positively charged carrier, or a premix thatmay in turn be used to produce such a formulation. Also provided arekits that contain means for sequentially administering a botulinum toxincomplex (or a reduced botulinum toxin complex including just the 150 KDneurotoxin itself or the neurotoxin with some native complex proteins)and a positively charged carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: A bar graph showing the required time to return to the baselineDAS value (0.4) following repeated administration of either RT003 orBOTOX®

FIG. 2: FIG. 2A shows the hind leg of a mouse injected with a dark dyeto indicate the portion of a mouse's gastrocnemius muscle that isaffected by lateral-to-midline injection. FIG. 2B shows the hind leg ofa mouse injected with a dark dye to indicate the portion of a mouse'sgastrocnemius muscle that is affected by midline injection.

FIG. 3: Digital abduction scores measured as a function of timefollowing injection of RT003, RTT150, or BOTOX® into either thelateral-to-midline or midline portion of the gastrocnemius muscle of amouse.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to novel injectable compositions comprisingbotulinum toxin, a botulinum toxin complex, or a reduced botulinum toxincomplex. In preferred embodiments, the compositions stabilize the toxinor enable the transport or delivery of toxin through tissues afterinjection such that the toxin has reduced antigenicity, a better safetyprofile, enhanced potency, faster onset of clinical efficacy and/orlonger duration of clinical efficacy compared to conventional commercialbotulinum toxin complexes that are bound to exogenous albumin (e.g.,BOTOX® or MYOBLOC®). The compositions of the invention may be used asinjectable applications for providing a botulinum toxin to a subject,for various therapeutic, aesthetic and/or cosmetic purposes, asdescribed herein. The compositions of the invention also have animproved safety profile over other compositions and methods of deliveryof botulinum toxin. In addition, these compositions can affordbeneficial reductions in immune responses to the botulinum toxin.

The term “botulinum toxin” as used herein may refer to any of the knowntypes of botulinum toxin (e.g., 150 kD botulinum toxin protein moleculesassociated with the different serotypes of C. botulinum), whetherproduced by the bacterium or by recombinant techniques, as well as anysuch types that may be subsequently discovered including newlydiscovered serotypes, and engineered variants or fusion proteins. Asmentioned above, currently seven immunologically distinct botulinumneurotoxins have been characterized, namely botulinum neurotoxinserotypes A, B, C, D, E, F and G, each of which is distinguished byneutralization with type-specific antibodies. The botulinum toxinserotypes are commercially available, for example, from Sigma-Aldrich(St. Louis, Mo.) and from Metabiologics, Inc. (Madison, Wis.), as wellas from other sources. The different serotypes of botulinum toxin varyin the animal species that they affect and in the severity and durationof the paralysis they evoke. At least two types of botulinum toxin,types A and B, are available commercially in formulations for treatmentof certain conditions. Type A, for example, is contained in preparationsof Allergan having the trademark BOTOX®^(M) and of Ipsen having thetrademark DYSPORT®, and type B is contained in preparations of Elanhaving the trademark MYOBLOC®.

The term “botulinum toxin” used in the compositions of this inventioncan alternatively refer to a botulinum toxin derivative, that is, acompound that has botulinum toxin activity but contains one or morechemical or functional alterations on any part or on any amino acidchain relative to naturally occurring or recombinant native botulinumtoxins. For instance, the botulinum toxin may be a modified neurotoxinthat is a neurotoxin which has at least one of its amino acids deleted,modified or replaced, as compared to a native form, or the modifiedneurotoxin can be a recombinantly produced neurotoxin or a derivative orfragment thereof. For instance, the botulinum toxin may be one that hasbeen modified in a way that, for instance, enhances its properties ordecreases undesirable side effects, but that still retains the desiredbotulinum toxin activity. Alternatively the botulinum toxin used in thisinvention may be a toxin prepared using recombinant or syntheticchemical techniques, e.g. a recombinant peptide, a fusion protein, or ahybrid neurotoxin, for example prepared from subunits or domains ofdifferent botulinum toxin serotypes (see U.S. Pat. No. 6,444,209, forinstance). The botulinum toxin may also be a portion of the overallmolecule that has been shown to possess the necessary botulinum toxinactivity, and in such case may be used per se or as part of acombination or conjugate molecule, for instance a fusion protein.Alternatively, the botulinum toxin may be in the form of a botulinumtoxin precursor, which may itself be non-toxic, for instance a non-toxiczinc protease that becomes toxic on proteolytic cleavage.

The term “botulinum toxin complex” or “toxin complex” as used hereinrefers to the approximately 150 kD botulinum toxin protein molecule(belonging to any one of botulinum toxin serotypes A-G), along withassociated endogenous non-toxin proteins (i.e., hemagglutinin proteinand non-toxin non-hemagglutinin protein produced by Clostridiumbotulinum bacteria). Note, however, that the botulinum toxin complexneed not be derived from Clostridium botulinum bacteria as one unitarytoxin complex. For example, botulinum toxin or modified botulinum toxinmay be recombinantly prepared first and then subsequently combined withthe non-toxin proteins. Recombinant botulinum toxin can also bepurchased (e.g., from List Biological Laboratories, Campbell, Ca.) andthen combined with non-toxin proteins.

This invention also contemplates modulation of the stability ofbotulinum toxin molecules through the addition of one or more exogenousstabilizers, the removal of endogenous stabilizers, or a combinationthereof. For example, this invention contemplates the use of “reducedbotulinum toxin complexes”, in which the botulinum toxin complexes havereduced amounts of non-toxin protein compared to the amounts naturallyfound in botulinum toxin complexes produced by Clostridium botulinumbacteria. In one embodiment, reduced botulinum toxin complexes areprepared using any conventional protein separation method to extract afraction of the hemagglutinin protein or non-toxin non-hemagglutininprotein from botulinum toxin complexes derived from Clostridiumbotulinum bacteria. For example, reduced botulinum toxin complexes maybe produced by dissociating botulinum toxin complexes through exposureto red blood cells at a pH of 7.3 (e.g., see EP 1514556 A1, herebyincorporated by reference). HPLC, dialysis, columns, centrifugation, andother methods for extracting proteins from proteins can be used.Alternatively, when the reduced botulinum toxin complexes are to beproduced by combining synthetically produced botulinum toxin withnon-toxin proteins, one may simply add less hemagglutinin or non-toxin,non-hemagglutinin protein to the mixture than what would be present fornaturally occurring botulinum toxin complexes. Any of the non-toxinproteins (e.g., hemagglutinin protein or non-toxin non-hemagglutininprotein or both) in the reduced botulinum toxin complexes according tothe invention may be reduced independently by any amount. In certainexemplary embodiments, one or more non-toxin proteins are reduced by atleast about 0.5%, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100% compared to the amounts normally found in botulinum toxincomplexes. As noted above, Clostridium botulinum bacteria produce sevendifferent serotypes of toxin and commercial preparations aremanufactured with different relative amounts of non-toxin proteins (i.e.different amount of toxin complexes). For example, MYOBLOC™ has 5000 Uof Botulinum toxin type B per ml with 0.05% human serum albumin, 0.01 Msodium succinate, and 0.1 M sodium chloride. DYSPORT™ has 500 U ofbotulinum toxin type A-hemagglutinin complex with 125 mcg albumin and2.4 mg lactose. In certain embodiments, substantially all of thenon-toxin protein (e.g., greater than 95%, 96%, 97%, 98% or 99% of thehemagglutinin protein and non-toxin non-hemagglutinin protein) thatwould normally be found in botulinum toxin complexes derived fromClostridium botulinum bacteria is removed from the botulinum toxincomplex. Furthermore, although the amount endogenous non-toxin proteinsmay be reduced by the same amount in some cases, this invention alsocontemplates reducing each of the endogenous non-toxin proteins bydifferent amounts, as well as reducing at least one of the endogenousnon-toxin proteins, but not the others.

As noted above, an exogenous stabilizer (e.g., albumin) is typicallyadded to stabilize botulinum toxin formulations. For instance, in thecase of BOTOX®, 0.5 mg of human albumin per 100 U of type A botulinumtoxin complex to stabilize the complex. Generally, the amount ofexogenous stabilizer that may be added to stabilize the compositionsaccording to the invention is not particularly limited. In someembodiments, the amount of added stabilizer may be less than the amountconventionally added, owing to the ability of positively chargedcarriers of the invention to act as a stabilizer in its own right. Forinstance, the amount of added exogenous albumin can be any amount lessthan the conventional thousand-fold excess of exogenous albumin and, incertain exemplary embodiments of the invention, is only about 0.25,0.20, 0.15, 0.10, 0.01, 0.005, 0.001, 0.0005, 0.00001, 0.000005,0.000001, or 0.0000001 mg per 100 U of botulinum toxin. In oneembodiment, no exogenous albumin is added as a stabilizer to thecompositions of the invention.

According to the present invention, a positively charged carriermolecule having protein transduction domains or efficiency groups, asdescribed herein, has been found suitable as a transport system for abotulinum toxin, enabling toxin to be injected with improved penetrationto target structures such as muscles and/or other skin-associatedstructures. The transport occurs without covalent modification of thebotulinum toxin. Besides enhancing penetration of botulinum toxin, thepositively charged carriers of the invention may, in certain preferredembodiments, stabilize the botulinum toxin against degradation. In suchembodiments, the hemagglutinin protein and non-toxin, non-hemagglutininprotein that are normally present to stabilize the botulinum toxin maybe reduced or omitted entirely. Similarly, the exogenous albumin that isnormally added during manufacturing may be omitted.

By the use of the terms “positively charged” or “cationic” in connectionwith the term “carrier”, it is meant that the carrier has a positivecharge under at least some solution-phase conditions, more preferablyunder at least some physiologically compatible conditions. Morespecifically, “positively charged” and “cationic” as used herein, meansthat the group in question contains functionalities that are chargedunder all pH conditions, for instance, a quaternary amine, or contains afunctionality which can acquire positive charge under certainsolution-phase conditions, such as pH changes in the case of primaryamines. More preferably, “positively charged” or “cationic” as usedherein refers to those groups that have the behavior of associating withanions over physiologically compatible conditions. Polymers with amultiplicity of positively-charged moieties need not be homopolymers, aswill be apparent to one skilled in the art. Other examples of positivelycharged moieties are well known in the prior art and can be employedreadily, as will be apparent to those skilled in the art.

Generally, the positively-charged carrier (also referred to as a“positively charged backbone”) is typically a chain of atoms, eitherwith groups in the chain carrying a positive charge at physiological pH,or with groups carrying a positive charge attached to side chainsextending from the backbone. In certain preferred embodiments, thepositively charged backbone is a cationic peptide. As used herein, theterm “peptide” refers to an amino acid sequence, but carries noconnotation with respect to the number of amino acid residues within theamino acid sequence. Accordingly, the term “peptide” may also encompasspolypeptides and proteins. In certain preferred embodiments, thepositively charged backbone itself will not have a defined enzymatic ortherapeutic biologic activity. In certain embodiments, the backbone is alinear hydrocarbon backbone which is, in some embodiments, interruptedby heteroatoms selected from nitrogen, oxygen, sulfur, silicon andphosphorus. The majority of backbone chain atoms are usually carbon.Additionally, the backbone will often be a polymer of repeating units(e.g., amino acids, poly(ethyleneoxy), poly(propyleneamine),polyalkyleneimine, and the like) but can be a heteropolymer. In onegroup of embodiments, the positively charged backbone is apolypropyleneamine wherein a number of the amine nitrogen atoms arepresent as ammonium groups (tetra-substituted) carrying a positivecharge. In another embodiment, the positively charged backbone is anonpeptidyl polymer, which may be a hetero- or homo-polymer such as apolyalkyleneimine, for example a polyethyleneimine orpolypropyleneimine, having a molecular weight of from about 10,000 toabout 2,500,000, preferably from about 100,000 to about 1,800,000, andmost preferably from about 500,000 to about 1,400,000. In another groupof embodiments, the backbone has attached a plurality of side-chainmoieties that include positively charged groups (e.g., ammonium groups,pyridinium groups, phosphonium groups, sulfonium groups, guanidiniumgroups, or amidinium groups). The sidechain moieties in this group ofembodiments can be placed at spacings along the backbone that areconsistent in separations or variable. Additionally, the length of thesidechains can be similar or dissimilar. For example, in one group ofembodiments, the sidechains can be linear or branched hydrocarbon chainshaving from one to twenty carbon atoms and terminating at the distal end(away from the backbone) in one of the above-noted positively chargedgroups. The association between the positively charged carrier and thebotulinum toxin, reduced bo is by non-covalent interaction, non-limitingexamples of which include ionic interactions, hydrogen bonding, van derWaals forces, or combinations thereof.

In one group of embodiments, the positively charged backbone is apolypeptide having multiple positively charged sidechain groups (e.g.,lysine, arginine, ornithine, homoarginine, and the like). Preferably,the polypeptide has a molecular weight from about 100 to about1,500,000, more preferably from about 500 to about 1,200,000, mostpreferably from about 1000 to about 1,000,000. One of skill in the artwill appreciate that when amino acids are used in this portion of theinvention, the sidechains can have either the D- or L-form (R or Sconfiguration) at the center of attachment. In certain preferredembodiments, the polypeptide has a molecular weight from about 500 toabout 5000, more preferably from 1000 to about 4000, more preferablyfrom 2000 to about 3000.

Alternatively, the backbone may comprise amino acid analogs and/orsynthetic amino acids. The backbone may also be an analog of apolypeptide such as a peptoid. See, for example, Kessler, Angew. Chem.Int. Ed. Engl. 32:543 (1993); Zuckermann et al. Chemtracts-Macromol.Chem. 4:80 (1992); and Simon et al. Proc. Nat'l. Acad. Sci. USA 89:9367(1992)). Briefly, a peptoid is a polyglycine in which the sidechain isattached to the backbone nitrogen atoms rather than the α-carbon atoms.As above, a portion of the sidechains will typically terminate in apositively charged group to provide a positively charged backbonecomponent. Synthesis of peptoids is described in, for example, U.S. Pat.No. 5,877,278, which is hereby incorporated by reference in itsentirety. As the term is used herein, positively charged backbones thathave a peptoid backbone construction are considered “non-peptide” asthey are not composed of amino acids having naturally occurringsidechains at the alpha-carbon locations.

A variety of other backbones can be used employing, for example, stericor electronic mimics of polypeptides wherein the amide linkages of thepeptide are replaced with surrogates such as ester linkages, thioamides(—CSNH—), reversed thioamide (—NHCS—), aminomethylene (—NHCH₂—) or thereversed methyleneamino (—CH₂NH—) groups, keto-methylene (—COCH₂—)groups, phosphinate (—PO₂RCH₂—), phosphonamidate and phosphonamidateester (—PO₂RNH—), reverse peptide (—NHCO—), trans-alkene (—CR═CH—),fluoroalkene (—CF═CH—), dimethylene (—CH₂CH₂—), thioether (—CH₂S—),hydroxyethylene (—CH(OH)CH₂—), methyleneoxy (—CH₂O—), tetrazole (CN₄),sulfonamido (—SO₂NH—), methylenesulfonamido (—CHRSO₂NH—), reversedsulfonamide (—NHSO₂—), and backbones with malonate and/orgem-diamino-alkyl subunits, for example, as reviewed by Fletcher et al.((1998) Chem. Rev. 98:763) and detailed by references cited therein.Many of the foregoing substitutions result in approximately isostericpolymer backbones relative to backbones formed from α-amino acids.

In each of the backbones provided above, sidechain groups can beappended that carry a positively charged group. For example, thesulfonamide-linked backbones (—SO₂NH— and —NHSO₂—) can have sidechaingroups attached to the nitrogen atoms. Similarly, the hydroxyethylene(—CH(OH)CH₂—) linkage can bear a sidechain group attached to the hydroxysubstituent. One of skill in the art can readily adapt the other linkagechemistries to provide positively charged sidechain groups usingstandard synthetic methods.

In one embodiment, the positively charged backbone is a polypeptidehaving protein transduction domains (also referred to as efficiencygroups). As used herein, an efficiency group or protein transductiondomain is any agent that has the effect of promoting the translocationof the positively charged backbone through a tissue or cell membrane.Non-limiting examples of protein transduction domains or efficiencygroups include -(gly)_(n1)-(arg)_(n2) (SEQ ID NO: 1), HIV-TAT orfragments thereof, or the protein transduction domain of Antennapedia,or a fragment thereof, in which the subscript n1 is an integer of from 0to 20, more preferably 0 to 8, still more preferably 2 to 5, and thesubscript n2 is independently an odd integer of from about 5 to about25, more preferably about 7 to about 17, most preferably about 7 toabout 13. In some embodiments, the HIV-TAT fragment does not contain thecysteine-rich region of the HIV-TAT molecule, in order to minimize theproblems associated with disulfide aggregation. Still further preferredare those embodiments in which the HIV-TAT fragment has the formula(gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 2),(gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 3) or(gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ ID NO: 4) wherein the subscripts pand q are each independently an integer of from 0 to 20 and the fragmentis attached to the backbone via either the C-terminus or the N-terminusof the fragment. In certain preferred embodiments, p is one and q iszero or p is zero and q is one. Preferred HIV-TAT fragments are those inwhich the subscripts p and q are each independently integers of from 0to 8, more preferably 0 to 5. In another preferred embodiment thepositively charged side chain or branching group is the Antennapedia(Antp) protein transduction domain (PTD), or a fragment thereof thatretains activity. These are known in the art, for instance, from Consoleet al., J. Biol. Chem. 278:35109 (2003) and a non-limiting example of aAntp PTD contemplated by this invention is SGRQIKIWFQNRRMKWKKC (SEQ IDNO: 5).

Preferably the positively charged carrier includes side-chain positivelycharged protein transduction domains in an amount of at least about0.01%, as a percentage of the total carrier weight, preferably fromabout 0.01 to about 50 weight percent, more preferably from about 0.05to about 45 weight percent, and most preferably from about 0.1 to about30 weight %. For positively charged protein transduction domains havingthe formula -(gly)_(n1)-(arg)_(n2) (SEQ ID NO: 1), a preferred range isfrom about 0.1 to about 25%.

In another embodiment, the backbone portion is a polylysine andpositively charged protein transduction domains are attached to thelysine sidechain amino groups or to the C- or N termini. In somepreferred embodiments, the polylysine may have a molecular weight thatis at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500,2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 D, and less thanabout 2,000,000, 1,000,000, 500,000, 250,000, 100,000, 75,000, 50,000,and 25,000 D. Within the range of 100 to 2,000,000 D, it is contemplatedthat the lower and/or upper range may be increased or decreased,respectively, by 100, with each resulting sub-range being a specificallycontemplated embodiment of the invention. In some exemplary embodiments,the polylysine has a molecular weight from about 1,000 to about1,500,000 D, from about 2,000 to about 800,000 D, or from about 3,000 toabout 200,000 D. In other exemplary embodiments, the polylysine hasmolecular weight from about 100 to about 10,000 D, from about 500 toabout 5,000 D, from about 1,000 to about 4,000 D, from about 1,500 toabout 3,500 D or from about 2,000 to about 3,000 D. In some embodiments,the polylysine contemplated by this invention can be any of thecommercially available (Sigma Chemical Company, St. Louis, Mo., USA)polylysines such as, for example, polylysine having MW>70,000,polylysine having MW of 70,000 to 150,000, polylysine having MW 150,000to 300,000 and polylysine having MW>300,000. The selection of anappropriate polylysine will depend on the remaining components of thecomposition and will be sufficient to provide an overall net positivecharge to the composition and provide a length that is preferably fromone to four times the combined length of the negatively chargedcomponents. Preferred positively charged protein transduction domains orefficiency groups include, for example,-gly-gly-gly-arg-arg-arg-arg-arg-arg-arg (-Gly₃Arg₇) (SEQ ID NO: 6) orHIV-TAT.

In another preferred embodiment the positively charged backbone is apolyalkyleneimine, non-limiting examples of which includepolyethyleneimine, polypropyleneimine, and polybutyleneimine. In certainembodiments, the polyalkyleneimine has a molecular weight of at least100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, or 6000 D, and less than about2,000,000, 1,000,000, 500,000, 250,000, 100,000, 75,000, 50,000, and25,000 D. Within the range of 100 to 2,000,000 D, it is contemplatedthat the lower and/or upper range may be increased or decreased,respectively, by 100, with each resulting sub-range being a specificallycontemplated embodiment of the invention.

In other embodiments of this invention, the carrier is a relativelyshort polylysine or polyethyleneimine (PEI) backbone (which may belinear or branched) and which has positively charged branching groups.Without wishing to be constrained by theory, it is believed that suchcarriers are useful for minimizing uncontrolled aggregation of thebackbones and botulinum toxin in a therapeutic composition, which causesthe transport efficiency to decrease dramatically. When the carrier is arelatively short linear polylysine or PEI backbone, the backbone willhave a molecular weight of less than 75,000 D, more preferably less than30,000 D, and most preferably, less than 25,000 D. When the carrier is arelatively short branched polylysine or PEI backbone, however, thebackbone will have a molecular weight less than 60,000 D, morepreferably less than 55,000 D, and most preferably less than 50,000 D.

In one particularly interesting embodiment, the non-native molecules arecationic peptides that have no inherent botulinum-toxin-like activityand that also contain one or more protein transduction domains asdescribed herein. Without wishing to be bound by any particularscientific theory, it is believed that the peptides enhance tissuepenetration of molecules associated in complex after injection, whileenhancing stabilization of the botulinum toxin in skin and in vitro. Itis believed that the enhanced tissue penetration afforded by thesepeptides in particular affords reduced antigenicity, a better safetyprofile, enhanced potency, faster onset of clinical efficacy or longerduration of clinical efficacy compared to conventional commercialbotulinum toxin complexes that are bound to exogenous albumin (e.g.,BOTOX® or MYOBLOC®).

In preferred embodiments, the concentration of positively chargedcarriers in the compositions according to the invention is sufficient toenhance the delivery of the botulinum toxin to molecular targets suchas, for example, motor nerve plates. Furthermore, without wishing to bebound by theory, it is believed that the penetration rate followsreceptor-mediated kinetics, such that tissue penetration increases withincreasing amounts of penetration-enhancing-molecules up to a saturationpoint, upon which the transport rate becomes constant. Thus, in apreferred embodiment, the amount of addedpenetration-enhancing-molecules is equal to the amount that maximizespenetration rate right before saturation. A useful concentration rangefor the positively charged carrier in the injectable compositions ofthis invention is about 0.1 pg to about 1.0 mg per unit of the botulinumtoxin composition as described herein. More preferably, the positivelycharged carrier in the topical compositions of the invention is presentin the range of about 1.0 pg to 0.5 mg per unit of botulinum toxin.

Compositions of this invention are preferably in a form that permitsinjection into the skin or epithelium of subjects or patients, (i.e.,humans or other mammals in need of the particular treatment). The term“in need” is meant to include both pharmaceutical or health-relatedneeds (e.g., treating conditions involving undesirable facial musclespasms), as well as cosmetic and subjective needs (e.g., altering orimproving the appearance of facial tissue). In preferred embodiments,the compositions are prepared by mixing the botulinum toxin (eithercontaining the associated non-toxin proteins or reduced associatednon-toxin proteins) with the positively charged carrier, and usuallywith one or more additional pharmaceutically acceptable carriers orexcipients. In their simplest form, they may contain an aqueouspharmaceutically acceptable diluent, such as buffered saline (e.g.,phosphate buffered saline). However, the compositions may contain otheringredients typically found in injectable pharmaceutical orcosmeceutical compositions, including a dermatologically orpharmaceutically acceptable carrier, vehicle or medium that iscompatible with the tissues to which it will be applied. The term“dermatologically or pharmaceutically acceptable,” as used herein, meansthat the compositions or components thereof so described are suitablefor use in contact with these tissues or for use in patients in generalwithout undue toxicity, incompatibility, instability, allergic response,and the like. As appropriate, compositions of the invention may compriseany ingredient conventionally used in the fields under consideration,and particularly in cosmetics and dermatology.

In terms of their form, compositions of this invention may includesolutions, emulsions (including microemulsions), suspensions, gels,powders, or other typical solid or liquid compositions used forinjection to muscle and other tissues where the compositions may beused. In preferred embodiments, the compositions of the invention arepresent in low-viscosity, sterile formulations suitable for injectionwith a syringe. The compositions of the invention may be in the form ofa lyophilized powder that is reconstituted using a pharmaceuticallyacceptable liquid diluent prior to injection. In certain embodiments,the lyophilized powder is reconstituted with a liquid diluent to form aninjectable formulation with a viscosity of about 0.1 to about 2000 cP,more preferably about 0.2 to about 500 cP, even more preferably about0.3 to about 50 cP, and even more preferably about 0.4 to about 2.0 cP.The compositions of the invention may contain, in addition to thebotulinum toxin and positively charged carrier, other ingredientstypically used in such products, such as antimicrobials, hydrationagents, tissue bulking agents or tissue fillers, preservatives,emulsifiers, natural or synthetic oils, solvents, surfactants,detergents, gelling agents, antioxidants, fillers, thickeners, powders,viscosity-controlling agents and water, and optionally includinganesthetics, anti-itch actives, botanical extracts, conditioning agents,minerals, polyphenols, silicones or derivatives thereof, vitamins, andphytomedicinals.

The injectable compositions according to this invention may be in theform of controlled-release or sustained-release compositions whichcomprise botulinum toxin and positively charged carrier encapsulated orotherwise contained within a material such that they are released withinthe tissue in a controlled manner over time. The composition comprisingthe botulinum toxin and positively charged carrier may be containedwithin matrixes, liposomes, vesicles, microcapsules, microspheres andthe like, or within a solid particulate material, all of which isselected and/or constructed to provide release of the botulinum toxinover time. The botulinum toxin and the positively charged carrier may beencapsulated together (i.e., in the same capsule) or separately (i.e.,in separate capsules).

Botulinum toxin formulations according to the invention can be deliveredby injection (typically using a syringe) to muscles underlying the skin,or to glandular structures within the skin, in an effective amount toproduce paralysis, produce relaxation, alleviate contractions, preventor alleviate spasms, reduce glandular output, or other desired effects.Local delivery of the botulinum toxin in this manner could afford dosagereductions, reduce toxicity and allow more precise dosage optimizationfor desired effects relative to injectable or implantable materials.

The compositions of the invention are administered to deliver aneffective amount of the botulinum toxin. The term “effective amount” asused herein means an amount of a botulinum toxin as defined above thatis sufficient to produce the desired muscular paralysis or otherbiological or aesthetic effect, but that implicitly is a safe amount,i.e. one that is low enough to avoid serious side effects. Desiredeffects include the relaxation of certain muscles with the aim of, forinstance, decreasing the appearance of fine lines and/or wrinkles,especially in the face, or adjusting facial appearance in other wayssuch as widening the eyes, lifting the corners of the mouth, orsmoothing lines that fan out from the upper lip, or the general reliefof muscular tension. The last-mentioned effect, general relief ofmuscular tension, can be effected in the face or elsewhere. Thecompositions of the invention may contain an appropriate effectiveamount of the botulinum toxin for application as a single-dosetreatment, or may be more concentrated, either for dilution at the placeof administration or for use in multiple applications. Through the useof the positively charged carrier this invention, a botulinum toxin canbe administered by injection to a subject for treating conditions suchas wrinkles, undesirable facial muscle or other muscular spasms,hyperhidrosis, acne, or conditions elsewhere in the body in which reliefof muscular ache or spasms is desired. The botulinum toxin isadministered by injection to muscles or to other skin-associated orother target tissue structures. The administration may be made, forexample, to the legs, shoulders, back (including lower back), axilla,palms, feet, neck, face, groin, dorsa of the hands or feet, elbows,upper arms, knees, upper legs, buttocks, torso, pelvis, or any otherparts of the body where administration of the botulinum toxin isdesired.

Administration of the injectable botulinum toxin-containing compositionsof this invention may also be carried out to treat other conditions,including any condition for which prevention of synaptic transmission ofor release of acetylcholine would confer a therapeutic benefit. Forexample, the conditions that may be treated by the compositionsaccording to the invention include, without limitation, neurologic pain,migraine headache or other headache pain, overactive bladder, rhinitis,sinusitis, acne, dystonia, dystonic contractions (whether subjective orclinical), hyperhidrosis (whether subjective or clinical), andhypersecretion of one or more glands controlled by the cholinergicnervous system. The compositions of this invention may also be used forreducing or enhancing immune response, or treatment of other conditionsfor which administration of botulinum toxin by injection has beensuggested or performed.

Most preferably, the compositions are administered by or under thedirection of a physician or other health care professional. They may beadministered in a single treatment or in a series of treatments overtime. In preferred embodiments, a composition according to the inventionis injected at a location or locations where an effect associated withbotulinum toxin is desired. Because of its nature, the botulinum toxinpreferably is administered at an amount, application rate, and frequencythat will produce the desired result without producing any adverse orundesired results. For instance, in certain embodiments, thecompositions of the invention are applied at a rate of from about 1 U toabout 20,000 U, and more preferably from about 1 U to about 10,000 Ubotulinum toxin per cm² of skin surface. Higher dosages within theseranges may be employed, for example, in situations where the botulinumtoxin is administered in conjunction with controlled release materials,as described herein. In certain embodiments, the botulinum toxinformulations of the invention are administered to provide 1 to 400 U,more preferably 10 to 350 U, even more preferably 30 to 250 U and mostpreferably 50 to 200 U of botulinum toxin per injection.

This invention also contemplates the use of a variety of deliverydevices for injecting botulinum toxin-containing compositions describedherein across skin. Such devices may include, without limitation, aneedle and syringe, or may involve more sophisticated devices capable ofdispensing and monitoring the dispensing of the composition, andoptionally monitoring the condition of the subject in one or moreaspects (e.g., monitoring the reaction of the subject to the substancesbeing dispensed).

In some embodiments, the compositions can be pre-formulated and/orpre-installed in a delivery device as such. This invention alsocontemplates embodiments wherein the compositions are provided in a kitthat stores one or more components separately from the remainingcomponents. For example, in certain embodiments, the invention providesfor a kit that separately stores botulinum toxin and the positivelycharged carrier for combining at or prior to the time of application.The amount of positively charged carrier or the concentration ratio ofthese molecules to the botulinum toxin will depend on which carrier ischosen for use in the composition in question. The appropriate amount orratio of carrier molecule in a given case can readily be determined, forexample, by conducting one or more experiments such as those describedbelow.

In general, the invention also contemplates a method for administeringbotulinum toxin (alternatively as botulinum toxin complexes or reducedbotulinum toxin complexes) to a subject or patient in need thereof, inwhich an effective amount of botulinum toxin is administered inconjunction with a positively charged carrier, as described herein. By“in conjunction with” it is meant that the two components (botulinumtoxin and positively charged carrier) are administered in a combinationprocedure, which may involve either combining them prior toadministration to a subject, or separately administering them, but in amanner such that they act together to provide the requisite delivery ofan effective amount of the therapeutic protein. For example, acomposition containing the positively charged carrier may first beadministered to the skin of the subject, followed by application a skinpatch, syringe, or other device containing the botulinum toxin. Thebotulinum toxin may be stored in dry form in a syringe or otherdispensing device and the positively charged carrier may be injectedbefore application of the toxin so that the two act together, resultingin the desired tissue penetration enhancement. In that sense, thus, thetwo substances (positively charged carrier and botulinum toxin) act incombination or perhaps interact to form a composition or combination insitu. Accordingly, the invention also includes a kit with a device fordispensing botulinum toxin and a liquid, gel, or the like that containsthe positively charged carrier, and that is suitable for injection tothe skin or target tissue of a subject. Kits for administering thecompositions of the inventions, either under direction of a health careprofessional or by the patient or subject, may also include a customapplicator suitable for that purpose.

The compositions of this invention are suitable for use in physiologicenvironments with pH ranging from about 4.5 to about 6.3, and may thushave such a pH. The compositions according to this invention may bestored either at room temperature or under refrigerated conditions.

It is understood that the following examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

Example 1 Duration of Local Muscle Paralysis in a Murine Model.

This example compares the duration of local muscle paralysis in miceinjected with either RT003 or BOTOX®. RT003 is an exemplary injectableformulation according to the invention that contains type A botulinumtoxin (purified to remove all endogenous non-toxin proteins) andpositively charged carrier with the sequence RKKRRQRRRG-(K)₁₅-GRKKRRQRRR(SEQ ID NO: 7). BOTOX® also contains type A botulinum toxin, butexogenous albumin is added to stabilize the type A botulinum toxinmolecule.

The muscle paralysis was measured using digit abduction score (DAS)assay as reported by Aoki, K. R. in “A comparison of the safety marginsof botulinum neurotoxin serotypes A, B, and F in mice”, Toxicon 2001;39(12):1815-1820. In the DAS assay, a mouse is briefly suspended by itstail to cause a characteristic startle response in which the mouseextends its hind limbs and abducts its hind digits. The extent to whichthe mouse is able to exhibit this startle response is scored on afive-point scale (from 0-4), with zero representing a normal startleresponse and four representing maximal reduction in digit abduction andleg extension. The scoring is done by an observer with no knowledge ofthe extent to which the subject mouse has been treated with neurotoxin.The baseline score using the DAS assay was determined to be 0.4 for anuntreated population of animals.

The study reported in this example involved ten animals (5 mice in RT003group and 5 mice in BOTOX® group). Each of the animals was injectedthree times with the respective botulinum toxin formulation (i.e., RT003or BOTOX®), with a 40-day period in between each dosing. Afterinjection, the number of days that all of the animals in each test groupwas above the 0.4 baseline of the DAS assay was counted. The results,shown in FIG. 1, indicate that the DAS assay score for the RT003-treatedgroup stayed above the 0.4 baseline value for 25, 22, and 21 days,following the first, second, and third treatment, respectively. Incontrast, the DAS assay score for the BOTOX®-treated group stayed abovethe 0.4 baseline value for 11, 8, and 11 days, following the first,second, and third treatment, respectively.

These DAS assay data indicate that local muscle paralysis caused by theRT003 formulation lasts approximately twice as long as the local muscleparalysis caused by BOTOX®. This result has important implications fortherapeutic uses of RT003 and other injectable botulinumtoxin-containing compounds according to the invention. In particular, byusing injectable compositions according to the invention, one cansignificantly reduce the frequency of follow-up injections required tomaintain a particular cosmetic or therapeutic effect caused by thebotulinum toxin. In turn, the reduced frequency of application canresult in better long-term efficacy, as the subject is less prone todevelop antibodies to the botulinum toxin.

Example 2

Injectable Botulinum Toxin Formulations with an Improved Safety Profile

Over the last few decades, botulinum toxin has found use as atherapeutic agent for treating a variety of conditions, includingwrinkles, hyperhidrosis, and muscle spasms. However, as botulinum toxinis the most potent naturally occurring toxin known to humans, improperadministration of the toxin can be extremely dangerous. For instance,accidental systemic delivery of botulinum toxin can lead to paralysis,difficulty breathing, and even death. Moreover, even if botulinum toxinwere properly delivered to a localized region of the body as a part of atherapeutic treatment, the toxin has a natural tendency to diffuse overtime, thereby increasing the risk of unwanted paralysis in other partsof the body. For example, when botulinum toxin is injected around theeyes to treat wrinkles, it may diffuse to the muscles that control themovement of the eyelids. If this happens, the eyelid muscles may becomepartially paralyzed, leading to a well known condition know as “eyeliddroop,” in which the eyelid is partially closed and interferes withnormal vision.

One aspect of this invention is to provide injectable botulinum toxinformulations with an improved safety profile compared to currentlyavailable commercial botulinum toxin formulations. In preferredembodiments, the injectable botulinum toxin formulations have a reducedtendency to diffuse after injection. In this way, certain preferredformulations of the invention permit more accurate delivery of botulinumtoxin, dramatically reducing unwanted side effects associated withuncontrolled local diffusion of botulinum toxin.

This example reports a comparative study of the tendency of botulinumtoxin in various formulations to diffuse following injection. The studyinvolved three botulinum toxin formulations: (1) BOTOX®; (2) RT003, abuffered and stabilized solution containing the 150 kD type A botulinumtoxin molecule non-covalently associated with a positively chargedcarrier having the formula RKKRRQRRRG-(K)₁₅-GRKKRRQRRR (SEQ ID NO: 7);and (3) RTT150, which is identical to the RT003 formulation, except thatis does not contain the positively charged carrier present in RT003.

The gastrocnemius muscle of each of the mice used in the study wasinjected with one of the aforementioned botulinum toxin formulations,either at the lateral-to-midline portion of the muscle (FIG. 2A), or atthe midline portion of the muscle (FIG. 2B). DAS assays were performedon each of the mice for four days after injection with the botulinumtoxin to determine whether the botulinum toxin of the respectiveformulation exhibited any tendency to diffuse from the gastrocnemiusmuscle toward the hind paws of the mouse. From the DAS assays, anydecreased ability of the test animals to abduct their hind digits wasinterpreted as an indication of botulinum toxin diffusion.

FIG. 3 shows the results of the DAS assays performed after injecting thetest animals with the different botulinum toxin formulations asdescribed above. Note that the digital abduction scores are grouped intotwo clusters, corresponding to whether the injection was at the midlineor the lateral-to-midline portion of the gastrocnemius muscle. Thegenerally lower DAS scores for midline injections, as compared to DASscores for lateral-to-midline injections, indicates that the degree ofparalysis in the hind paws of the test animals is generally lessfollowing midline injection. Without wishing to be limited by theory, itis believed that this behavior results from the greater distance thatbotulinum toxin has to travel to reach the hind digits of a test animalfollowing midline injection, as compared to lateral-to-midlineinjection. This greater required distance of travel by the botulinumtoxin is believed to decrease the likelihood of paralysis of the hinddigits.

FIG. 3 shows a digital abduction score of zero for all four daysfollowing midline injection of the RT003 formulation. This resultindicates that the botulinum toxin in the RT003 formulation stayslocalized in the midline portion of the gastrocnemius muscle uponinjection and that no paralysis-causing diffusion occurs on thetimescale of the experiment. By contrast, digital abduction scores abovethe 0.4 DAS baseline are observed following injection of the RTT150 andBOTOX® formulations, with the average DAS score being higher for theBOTOX® formulation. The DAS results for the RTT150 and BOTOX®formulations indicate that hind digit paralysis of the test animals wasobserved after midline injection of these formulations, with a greaterdegree of paralysis observed after the injection of the BOTOX®formulation. These data suggest that the botulinum toxin molecules inthe RTT150 and BOTOX® formulations are capable of locally diffusingafter injection, with a greater degree of local diffusion for thebotulinum toxin molecules in the BOTOX® formulation.

FIG. 3 also shows that hind digit paralysis is observed for all testanimals following lateral-to-midline injection, irrespective of thespecific botulinum toxin formulation. As discussed above, this greaterdegree of paralysis following lateral-to-midline injection, as comparedto midline injection, is believed to relate to a shorter travel distancefor the botulinum toxin to the hind paws of the test animals. However,while all three botulinum toxin formulations exhibit paralysis-causingdiffusion following lateral-to-midline injection, the degree ofparalysis in test animals injected with RT003 is less, on average, thanthe degree of paralysis observed for the RTT150 and BOTOX® formulationsduring the timescale of the experiment. Thus, the DAS assay datacorresponding to lateral-to-midline injection is qualitatively similarto that for midline injection in that it shows a decreased tendency forlocal diffusion of botulinum toxin for the RT003 formulation, ascompared to RTT150 and BOTOX®.

A comparison of the local diffusion rate following midline injection andlateral-to-midline injection can be made by considering a parametercalled the “diffusion index”, which is defined according to Equation(1):

$\begin{matrix}{{{diffusion}\mspace{14mu} {index}} = {\frac{{midline}\mspace{14mu} {digital}\mspace{14mu} {abduction}\mspace{14mu} {score}}{{lateral}\text{-}{to}\text{-}{midline}\mspace{14mu} {digital}\mspace{14mu} {abduction}\mspace{14mu} {score}} \times 100.}} & (1)\end{matrix}$

Since digital abduction scores can range from 0 to 4, andlateral-to-midline digital abduction scores are expected to be higherthan midline digital abduction scores (as discussed above), diffusionindex values will typically range from 0 to 100. A diffusion index valuethat approaches 100 indicates that the ratio of the midline andlateral-to-midline digital abduction scores approaches unity. This mayoccur if the rates of diffusion following injection are sufficientlyhigh that the diffusion times for the botulinum toxin to reach and toparalyze the hind digits of the test animal following midline andlateral-to-midline injection are comparable or nearly the same. At theother extreme, diffusion index values that approach zero indicate thatthe ratio of the midline and lateral-to-midline digital abduction scoresis approaching zero. This may occur if diffusion of the botulinum toxinfollowing midline injection is so low that it is insufficient to causeparalysis in the hind digits of the test animals, even though paralysisis observed following lateral-to-midline injection.

Table 1 shows diffusion index values calculated using digital abductionscores following midline or lateral-to-midline injection of BOTOX®,RT003, and RTT150, as reported in the experiment corresponding to FIG.3. On the timescale of the experiment, the diffusion index valuescorresponding to injection of the BOTOX® formulation are higher than thevalues observed for the RTT150 and RT003 formulations. This indicatesthat, for injection of the BOTOX® formulation, the ratio of the midlineand lateral-to-midline digital abduction scores are closer to unity,compared to the ratios observed for the RTT150 and RT003 formulations.Since botulinum toxin must diffuse further to cause hind-digit paralysisof a test animal following midline injection, the observation that theratio of the midline and lateral-to-midline digital abduction scoresfollowing BOTOX® injection is closer to unity suggests that thebotulinum toxin diffusion rate following midline injection of BOTOX® isfairly substantial relative to the rate following lateral-to-midlineinjection. In other words, the increased diffusion path lengthassociated with midline injection is less of a barrier to causinghind-digit paralysis.

In contrast, the diffusion index values for RT003 are all zero on thefour-day timescale of the experiment. This result indicates that noparalysis-inducing diffusion is observed following midline injection ofRT003. In other words, the RT003 formulation, which contains the type Abotulinum toxin molecule non-covalently associated with a positivelycharged carrier, permits enhanced localization injected type A botulinumtoxin. In this way, the RT003 formulation affords an improved safetyprofile compared to that of the BOTOX® formulation and minimizesunwanted paralysis.

The observed diffusion index values for RTT150, while not zero as in thecase of RT003, are still less than those observed for the BOTOX®formulation [see Table 1]. This result indicates that enough botulinumtoxin diffusion occurs to produce observable hind digit paralysis on thefour-day timescale of the experiment, but that the time required forparalysis-causing diffusion of botulinum toxin is relatively longerfollowing midline injection.

TABLE 1 Botulinum toxin diffusion index measurements for RTT150, BOTOX ®and RT003. Days Post Treatment 0 1 2 3 4 BOTOX ® NA 42 38 38 9 RT003 NA0 0 0 0 RTT150 NA 20 20 27 17

Example 3

Injectable Botulinum Toxin Formulations with Reduced Tendency toGenerate Antibodies

When botulinum toxin is periodically injected into a patient to treat anunwanted condition such as wrinkles, it is often observed that efficacyof the botulinum toxin decreases with successive injections, even thoughthe duration of the effects of the botulinum toxin may remain the same.This phenomenon is believed to be the result of the formation ofantibodies to the botulinum toxin by the immune system of the patient.From a treatment perspective, the formation of antibodies to botulinumtoxin by the patient is undesirable, because increasingly larger dosesof botulinum toxin are then required to achieve the same effect, whichpresents serious issues related to both safety and cost.

In certain embodiments, this invention provides injectable botulinumtoxin formulations that have a decreased tendency to induce antibodyformation, as compared to currently available commercial injectablebotulinum toxin formulations. Thus, in these embodiments, botulinumtoxin formulations help to minimize the risk associated with botulinumtoxin injection by permitting one, over time, to use less toxin toachieve the same effect.

In this example, the DAS assay data obtained after repeated RT003 andBOTOX® injections as described in Example 2 are analyzed as a functionof time to determine how the efficacy of these two formulations changesupon repeated administration to the same test animals. Generally, afterrepeated administration of either formulation, the duration of effectsassociated with botulinum toxin were the same. However, the degree ofmuscle paralysis upon repeated administration varied depending on theformulation. To quantify the change in the degree of muscle paralysis,the percent change in the digital abduction scores following injectionof either RT003 or BOTOX® was determined according to Equation (2):

$\begin{matrix}{{\% \mspace{14mu} {change}\mspace{14mu} {in}\mspace{14mu} {DAS}} = {\frac{{{DAS}\mspace{14mu} {for}\mspace{14mu} {nth}\mspace{14mu} {treatment}} - {{DAS}\mspace{14mu} {for}\mspace{14mu} {first}\mspace{14mu} {treatment}}}{{DAS}\mspace{14mu} {for}\mspace{14mu} {first}\mspace{14mu} {treatment}} \times 100\%}} & (2)\end{matrix}$

Since the numerator of Equation (2) is the difference between themeasured digital abduction scores for the n^(th) and the firsttreatment, the percent change in DAS will be negative if the digitalabduction score measured for the n^(th) treatment is less than thedigital abduction score measured for the first treatment. In otherwords, the percent change in DAS is negative when less paralysis isobserved after the nth treatment, as compared to the first treatment.Table 2 shows the percent change in the measured DAS values followingrepeated administration of RT003 and BOTOX® formulations according tothe procedure described in Example 2.

TABLE 2 Percent Change in DAS Value after Repeated Administration ofRT003 and BOTOX ® 1st 1st 2nd treatment retreatment retreatment RT003 0% 0% −30% BOTOX ® 0% −44% −67%

As indicated in Table 2, after the first retreatment, the percent changein the digital abduction score was −44% for the BOTOX® formulation,which suggests a substantial drop in the efficacy. In contrast, thepercent change in the digital abduction score for the RT003 formulationwas zero, indicating that the DAS score after the second retreatment wasthe same as after the initial administration and first retreatment. Thisresult indicates that the degree of paralysis observed after the firstretreatment of RT003 is the same as the degree of paralysis followingthe first treatment and that negligible formation of neutralizingantibodies occurred in the test animals even after the firstretreatment. After the 2nd retreatment of RT003 and BOTOX®, thecalculated percent changes in DAS values were negative for bothformulations, although the magnitude of the percent change in DAS valuesfor the RT003 formulation was half of the value determined for BOTOX®.The larger and negative percent change in DAS values observed for BOTOX®suggest that the test animals had a higher rate of antibody generationto BOTOX®, as compared to RT003. Thus, these data indicate thatformulations contemplated by this invention, such as RT003, may have alower tendency to induce the formation of antibodies that neutralize theeffect of botulinum toxin. Accordingly, this result suggests that byusing formulations contemplated by this invention, one can, over time,use less botulinum toxin to achieve the same therapeutic effect.

Example 4

Injectable Botulinum Toxin Formulations with Improved Stability

This example demonstrates that the positively charged carrier moleculesused in the injectable botulinum toxin formulations of the invention notonly enhance the safety profile of the formulations (Example 2), butalso improve their stability. Table 3 shows the results of agingexperiments wherein the RT003 and RTT150 formulations are aged at 4° C.(RT003 only) and at 40° C. (both RT003 and RTT150) for various timeintervals. After aging at the specified temperatures for the specifiedtimes, the potency of the RT003 and RTT150 formulations were measuredvia a series of mouse IP LD50 assays. The results, summarized in Table3, indicate that the potency of RT003 is essentially unchanged followingaging at 4° C., even after six months. Furthermore, the potency of theRT003 formulation, as measured by the formulation's ability to kill thetarget animals in a mouse IP LD50 assay, decreases only slightly even ifthe RT003 formulation is aged at elevated temperature (40° C.) for sixmonths. By contrast, the RTT150 formulation exhibited a significantdecrease in potency following only one month of aging at 40° C. Sincethe RT003 and RTT150 formulations are identical, with the exception thatthe RT003 formulation also contains a positively charged carriermolecule having the formula RKKRRQRRRG-(K)₁₅-GRKKRRQRRR (SEQ ID NO: 7),these data indicate that the positively charged carrier moleculeimproves the stability of the botulinum toxin in the RT003 formulation.

TABLE 3 Results of Mouse IP LD50 Assays following Aging of RT003 andRTT150 At Various Conditions Condition Time (° C.) (months) % TargetRT003 4 0 100% 4 6 118% 40 6  93% RTT150 40 1 <50%

What is claimed is:
 1. A kit for providing a sterile injectablebotulinum toxin for administration by injection to achieve a therapeuticor cosmetic effect in a subject in need thereof, the kit comprising: abotulinum toxin component present in an amount suitable for injection,the botulinum toxin component selected from the group consisting of abotulinum toxin complex, reduced botulinum toxin complex, and botulinumtoxin, and a positively charged carrier comprising the amino acidsequence RKKRRQRRRG-(K)₁₅-GRKKRRQRRR, wherein the positively chargedcarrier is non-covalently associated with the botulinum toxin component;a pharmaceutically acceptable diluent suitable for injection; and adelivery device for injecting said botulinum toxin component.
 2. The kitaccording to claim 1, wherein the botulinum toxin component is obtainedfrom serotypes A, B, C, D, E, F, or G of C. botulinum.
 3. The kitaccording to claim 2, wherein the botulinum toxin component is a type Abotulinum toxin component.
 4. The kit according to claim 3, wherein saiddelivery device is a syringe and a needle.
 5. The kit according to claim3, wherein the botulinum toxin component and the positively chargedcarrier are pre-formulated in said kit.
 6. The kit according to claim 5,wherein the pre-formulated botulinum toxin component and positivelycharged carrier are provided separately from said pharmaceuticallyacceptable diluent in said kit.
 7. The kit according to claim 3, whereinthe botulinum toxin component is provided separately from the positivelycharged carrier in said kit.
 8. The kit according to claim 3, whereinthe therapeutic or cosmetic effect results from local muscle paralysisfollowing injection of said botulinum toxin component, said positivelycharged carrier, and said pharmaceutically acceptable diluent, andwherein said local muscle paralysis lasts longer than local muscleparalysis following injection of the same dose of botulinum toxin unitsfrom a formulation of 100 U of type A botulinum toxin with accessoryprotein, 0.5 mg of human albumin, and 0.9 mg of sodium chloride.
 9. Thekit according to claim 8, wherein said muscle paralysis lastsapproximately twice as long as muscle paralysis following injection ofthe same dose of botulinum toxin units from a formulation of 100 U oftype A botulinum toxin with accessory protein, 0.5 mg of human albumin,and 0.9 mg of sodium chloride.
 10. The kit according to claim 3, whereinantibody generation following injection of said botulinum toxincomponent, said positively charged carrier, and said pharmaceuticallyacceptable diluent into the subject's body is reduced compared toantibody generation following injection of the same dose of botulinumtoxin units from a formulation of 100 U of type A botulinum toxin withaccessory protein, 0.5 mg of human albumin, and 0.9 mg of sodiumchloride.
 11. The kit according to claim 3, wherein the positivelycharged carrier reduces local diffusion of the botulinum toxin componentfollowing injection.
 12. The kit according to claim 3, wherein thepositively charged carrier stabilizes the botulinum toxin componentagainst degradation.
 13. The kit according to claim 12, wherein thepositively charged carrier stabilizes the botulinum toxin componentagainst degradation to retain potency after 6 months aging at 4° C. 14.The kit according to claim 3, wherein the therapeutic effect is reducingor enhancing immune response or reducing a symptom associated with adisorder selected from the group consisting of muscular tension,muscular spasm, hemifacial spasm, adult onset spasmodic torticollis,anal fissure, blepharospasm, cerebral palsy, headache, migraineheadache, strabismus, temporomandibular joint disorder, neurologic pain,overactive bladder, rhinitis, sinusitis, acne, dystonia, dystoniccontractions, cervical dystonia, hyperhidrosis, and hypersecretion of agland controlled by the cholinergic nervous system.
 15. The kitaccording to claim 3, wherein the cosmetic effect is selected from thegroup consisting of decreasing the appearance of fine lines and/orwrinkles, adjusting facial appearance in a desirable way, widening theeyes, lifting corners of the mouth, and smoothing lines that fan outfrom the upper lip.
 16. The kit according to claim 3, wherein saidbotulinum toxin component is present in a single dose amount.
 17. Thekit according to claim 3, wherein said botulinum toxin component ispresent in an amount from 10 to 350 U.
 18. The kit according to claim17, wherein said botulinum toxin component is present in an amount from30 to 250 U.
 19. The kit according to claim 3, wherein said botulinumtoxin component is present in an amount from 10 to 200 U.
 20. The kitaccording to claim 19, wherein said botulinum toxin component is presentin said composition in an amount from 30 to 200 U.